Process for production and purification of recombinant lysosomal alpha-mannosidase

ABSTRACT

The present invention relates to a process for purification of recombinant alpha-mannosidase, a process for production of alpha-mannosidase, a composition comprising alpha-mannosidase, use of the composition as a medicament, use as a medicament for the treatment of alpha-mannosidosis and a method of treating alpha-mannosidosis and/or alleviating the symptoms of alpha-mannosidosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority to pending U.S.application Ser. No. 13/576,258, filed on Jul. 31, 2012 which is a U.S.National Phase application of PCT International Application NumberPCT/DK2011/050054, filed on Feb. 23, 2011, designating the United Statesof America and published in the English language, which is anInternational application of and claims the benefit of priority to U.S.Provisional Application No. 61/307,587, filed on Feb. 24, 2010, andDanish Patent Application No. PA 2010 70067, filed on Feb. 24, 2010. Thedisclosures of the above-referenced applications are hereby expresslyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a process for purification ofrecombinant alpha-mannosidase, a process for production ofalpha-mannosidase, a composition comprising alpha-mannosidase, use ofthe composition as a medicament, use as a medicament for the treatmentof alpha-mannosidosis and a method of treating alpha-mannosidosis and/oralleviating the symptoms of alpha-mannosidosis.

Alpha-Mannosidosis

Alpha-mannosidosis is a recessive, autosomal disease that occurs worldwide with a frequency of between 1/1,000,000 and 1/500,000. Mannosidosisis found in all ethnic groups in Europe, America, Africa and also Asia.It is detected in all countries with a good diagnostic service forlysosomal storage disorders, at a similar frequency. They are bornapparently healthy, however the symptoms of the diseases areprogressive. Alpha-mannosidosis displays clinical heterogeneity, rangingfrom very serious to very mild forms. Typical clinical symptoms are:mental retardation, skeletal changes, impaired immune system resultingin recurrent infections, hearing impairment and often the disease isassociated with a typical facial characteristics such as a coarse face,a prominent forehead, a flattened nasal bridge, a small nose, and abroad mouth. In the most severe cases (mannosidosis type I) the childrensuffer from hepatosplenomegaly, and they die during the first years oflife. Possibly this early death is caused by severe infections due tothe immunodeficiency caused by the disease. In milder cases(mannosidosis type 2) the patients usually reach adult age. The skeletalweaknesses of the patients result in the needs of wheeling chairs at age20 to 40. The disease causes a diffuse dysfunction of the brain oftenresulting in weak mental performances that excludes anything but themost basic skills of simple reading and writing. These problemsassociated with hearing inabilities and other clinical manifestationspreclude the patient from an independent life, the consequence beingthat lifelong caretaking is needed.

Lysosomal Alpha-Mannosidase

Alpha-mannosidosis results from a deficient activity of lysosomalalpha-mannosidase (LAMAN, EC3.2.1.24). The disease is characterised bymassive intracellular accumulation of mannose-rich oligosaccharides,that is oligosaccharides carrying α1,2-, α1,3- and α1,6-mannosylresidues at their non-reducing termini. These oligosaccharides mainlyoriginate from the intralysosomal degradation of glycoproteins withN-linked oligosaccharides. However, some originate from the catabolismof dolichol-linked oligosaccharides and from misfolded glycoproteinsredirected to the cytosol for degradation by the proteasome (Hirsch etal. EMBO J. 22, 1036-1046, 2003 and Saint-Pol et al. J. Biol. Chem. 274,13547-13555, 1999). The lysosomal storage is observed in a wide range ofcell types and tissues, including neurons in all brain regions. LAMAN isan exoglycosidase which hydrolyses these terminal, non-reducingalpha-D-mannose residues in alpha-D-mannosides from the non-reducing endduring the ordered degradation of the N-linked glycoproteins (Aronsonand Kuranda FASEB J 3:2615-2622. 1989). The human precursor enzyme issynthesised as a single polypeptide of 1011 amino acids including asignal peptide of 49 residues. The precursor is proteolyticallyprocessed into three main glycopeptides of 15, 42, and 70 kD to thematured enzyme in the lysosome. The 70 kD glycopeptide is furtherprocessed into three subunits linked by disulfide bridges. (Berg et al.Mol. Gen. and Metabolism 73, 18-29, 2001, Nilssen et al. Hum. Mol.Genet. 6, 717-726. 1997).

The Lysosomal Alpha-Mannosidase Gene

The gene coding for LAMAN (MANB) is located at chromosome 19(19cen-q12), (Kaneda et al. Chromosoma 95:8-12. 1987). MANB consists of24 exons, spanning 21.5 kb (GenBank accession numbers U60885-U60899;Riise et al. Genomics 42:200-207, 1997). The LAMAN transcript is >>3,500nucleotides (nts) and contains an open reading frame encoding 1,011amino acids (GenBank U60266.1). The cloning and sequencing of the humancDNA encoding LAMAN has been published in three papers (Nilssen et al.Hum. Mol. Genet. 6, 717-726. 1997; Liao et al. J. Biol. Chem. 271,28348-28358. 1996; Nebes et al. Biochem. Biophys. Res. Commun. 200,239-245. 1994). Curiously, the three sequences are not identical. Whencompared to the sequence of Nilssen et al. (accession # U60266.1) a TAto AT change at positions 1670 and 1671 resulting in a valine toasparitic acid substitution was found by Liao et al. and Nebes et al.Also a C to A change in pos 1152 was found which do not result in anychanges in the amino acid sequence.

Diagnosis

The diagnosis of alpha-mannosidosis is currently is based on clinicalevaluation, detection of mannose-rich oligosaccharides in urine, anddirect measurements of alpha-mannosidase activity in various cell types,such as leukocytes, fibroblasts, and amniocytes (Chester et al., In:Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism.Edi-Ermes, Milan, pp 89-120. 1982; Thomas and Beaudet. In: Scriver C R,Beaudet A L, Sly W A, Valle D (eds). The metabolic and molecular basesof inherited disease. Vol 5. McGraw-Hill, New York, pp 2529-2562. 1995).

Because the symptoms initially often are mild and the biochemicaldiagnosis is difficult, the diagnosis is frequently made late in thecourse of the disease. It is obvious that patients and their familieswould benefit substantially from an early diagnosis.

Animal Models

Alpha mannosidosis has been described in cattle (Hocking et al. BiochemJ 128:69-78. 1972), cats (Walkley et al. Proc. Nat. Acad. Sci. 91:2970-2974, 1994), and guinea pigs (Crawley et al. Pediatr Res 46:501-509, 1999). A mouse model was recently generated by targeteddisruption of the alpha-mannosidase gene (Stinchi et al. Hum Mol Genet8: 1366-72, 1999) Like in humans alpha mannosidase seems to be caused byspecific mutations in the gene coding for lysosomal alpha-mannosidase.Berg et al. (Biochem J. 328:863-870.1997) reported the purification offeline liver lysosomal alpha-mannosidase and determination of its cDNAsequence. The active enzyme consists of 3 polypeptides, with molecularmasses reported to be 72, 41, and 12 kD. Similarly to the human enzymeit was demonstrated that the feline enzyme is synthesized as asingle-chain precursor with a putative signal peptide of 50 amino acidsfollowed by a polypeptide chain of 957 amino acids, which is cleavedinto the 3 polypeptides of the mature enzyme. The deduced amino acidsequence was 81.1% and 83.2% identical with the human and bovinesequences, respectively. A 4-bp deletion was identified in an affectedPersian cat; the deletion resulted in a frameshift from codon 583 andpremature termination at codon 645. No enzyme activity could be detectedin the liver of the cat. A domestic long-haired cat expressing a milderphenotype had enzyme activity of 2% of normal; this cat did not possessthe 4-bp deletion. Tollersrud et al. (Eur J Biochem 246:410-419. 1997)purified the bovine kidney enzyme to homogeneity and cloned the gene.The gene was organized in 24 exons that spanned 16 kb. Based on the genesequence they identified two mutations in cattle.

Medical Need for Alpha-Mannosidosis Therapy

In light of the severe clinical manifestations resulting from theaccumulation of mannose-rich oligosaccharides, the lack of effectivetreatment for alpha-mannosidosis is well recognised. At present, themajor therapeutic option for treatment of the disease is bone marrowtransplantation, however, it is the aim of the present invention topromote enzyme replacement therapy as a potential future alternative.

Bone Marrow Transplantation

In 1996 Walkley et al. (Proc. Nat. Acad. Sci. 91: 2970-2974, 1994)published a paper on three kittens with mannosisdosis that were treatedwith bone marrow transplantation (BMT) in 1991. In the 2 animals thatwere sacrificed a normalisation was seen, not only in the body, but moreimportantly, also in brain. The third cat was well after 6 years.Normally, an untreated cat dies with 3-6 months. In 1987 a child withmannosidosis was treated with BMT (Will et al. Arch Dis Child 1987October; 62(10):1044-9). He died after 18 weeks due to procedure relatedcomplications. In brain little enzyme activity was found. Thisdisappointing result could be explained by heavy immunosuppressivetreatment before death, or that it takes time for the enzyme activity toincrease in brain after BMT. The donor was the mother (who as carriermust be expected to have less than 50% enzyme activity) or it may be BMTin man has no effect on enzyme function in brain. Despite havingvariable outcomes the few attempts of bone marrow transplantation havethus indicated that successful engraftment can correct the clinicalmanifestations of alpha-mannosidosis, at least in part. However, thechallenge of reducing the serious procedure related complications whenapplying bone marrow transplantation in human therapy still remains tobe defeated.

Enzyme Replacement Therapy

When lysosomal storage diseases were discovered, hopes were raised thatthis could be treated by enzyme substitution. Enzyme replacement therapyhas proven efficient in Gaucher disease. When exogenous lysosomalglucocerebrosidase is injected into the patient, this enzyme is taken upby enzyme-deficient cells (Barton et al. N Engl J Med 324:1464-1470).Such uptake is regulated by certain receptors on the cell surface as forinstance the mannose-6-phosphate receptor, which is nearly ubiquitous onthe surface of cells and other receptors such as the asialoglycoproteinreceptor and the mannose receptor, which are restricted to certain celltypes such as cells of the monocyte/macrophage cell line andhepatocytes. The cellular uptake of the enzyme is therefore heavilydependent upon its glycosylation profile. If properly designed, thedeficient enzyme could be replaced by regular injections of exogenousenzyme in the same manner as diabetic patients receive insulin. In vitrostudies with the purified active lysosomal alpha-mannosidase added tothe media of enzyme-deficient fibroblasts showed correction of thelysosomal substrate accumulation. In vivo treatment, on the other hand,has been hampered in part by the problem of producing the sufficientquantity of enzymes, due to difficult large scale production andpurification procedures, and by complications resulting from immunereactions against the exogenous enzyme. Most importantly, however,special considerations apply in relation to lysosomal storage diseaseswith a major neurological component, such as alpha-mannosidosis, whereinthe clinical manifestations are related to increased lysosomal storagewithin the central nervous system. Thus, enzyme replacement therapy hasnot proven effective against the acute neuronopathic variant of Gaucherdisease (Prows et al. Am J Med Genet 71:16-21). The delivery oftherapeutic enzymes to the brain is prevented by the absence oftransport of these large molecules through the blood-brain barrier. Fromthe general notion that the blood brain barrier must be circumvented inorder to see an effect of therapeutic agents in the brain, the use of alarge diversity of delivery systems have been contemplated. Theseinclude invasive techniques such as osmotic opening of the blood brainbarrier with for instance mannitol and non-invasive techniques such asreceptor mediated endocytosis of chimeric enzymes. As enzyme replacementis expected to require administration of the enzyme on a regular basis,the use of invasive techniques should be avoided. Use of thenon-invasive techniques, has only recently provided promising results inanimal models (for alpha-mannosidosis see below, for other lysosomaldisorders see for example: Grubb et al. PNAS 2008, 105(7) pp.2616-2621). It has been contemplated that reduced storage in visceralorgans and in the meninges could reduce the amount of oligosaccharidesthat is carried to the brain. Such considerations, however, are notconsidered to be applicable to lysosomal disorders in which theneurological damage is primary and severe (Neufeld, E. F. Enzymereplacement therapy, in “Lysosomal disorders of the brain” (Platt, F. M.Walkley, S. V: eds Oxford University Press).

However, as described in Roces et al. Human Molecular Genetics 2004,13(18) pp. 1979-1988, Blanz et al. Human Molecular Genetics 2008, 17(22)pp. 3437-3445 and WO 05/094874 it has proven possible to increase levelsof LAMAN in the central nervous system of animals using e.g. intravenousinjection of a formulation comprising alpha-mannosidase thereby reducingintracellular levels of neutral mannose-rich oligosaccharides within oneor more regions of the central nervous system. This indicates thatrecombinant alpha-mannosidase is useful in enzyme replacement therapy ofpatients suffering from alpha-mannosidosis. Thus, one major remaininghurdle towards providing efficient treatment of alpha-mannosidosis usingenzyme replacement is providing sufficient amounts of pure recombinantalpha-mannosidase in a cost-efficient manner.

Production and Purification of Alpha-Mannosidase

WO 02/099092 discloses a small scale production process for rhLAMAN inCHO cells using serum free medium at 37° C. A small scale purificationprocess is also described involving diafiltration of the crude enzymeand weak anion exchange chromatography using DEAE sepharose FF columnsin the capture step, followed by a number of chromatographicpurification steps involving hydrophobic interaction- and mixed modechromatography. WO 05/094874 discloses a small scale production processfor rhLAMAN in Chinese Hamster Ovary (CHO) cells using serum free mediumat 37° C. A small scale purification process analogous to the one of WO02/099092 is also described. WO 05/077093 describes the manufacture ofhighly phosphorylated lysozymal enzymes. In example IV a purificationmethod for acid alpha-glucosidase (GAA) using a multi-modal resin(blue-sepharose) is described. GAA, although a lysozymal enzyme, ishowever entirely different from rhLAMAN. GAA is highly phosphorylated,while rhLAMAN has a low degree of phosphorylation. Furthermore, thesequence identity score is less than 12% between GAA and rhLAMAN, andfinally their theoretical isoelectric points differ by more than one pHunit (5.42 and 6.48 respectively). Thus the method as described in WO05/077093 to purify GAA is not applicable to rhLAMAN. A small scaleproduction process for rhLAMAN in CHO cells using 0.25% (V/V) serum andDMSO addition has been disclosed (Berg et al. Molecular Genetics andMetabolism, 73, pp 18-29, 2001. It also describes two purificationprocesses involving a) a three-step procedure involving ultrafiltration,anion exchange chromatography and gel filtration or b) single-stepimmuno-affinity chromatography. It is further disclosed how method a)results in the 130 kDa enzyme fragmenting entirely into 55 kDa and 72kDa fragments, whereas method b) results in partial fragmentation of the130 kDa precursor into significant amounts of the 55 and 72 kDafragments.

Hence, an improved process for production and purification ofrecombinant alpha-mannosidase would be advantageous. In particular, animproved process for large scale cultivation of a cell line capable ofexpressing alpha-mannosidase and a more efficient large scalepurification process for isolating pure alpha-mannosidase with a highenzymatic activity from a cell culture would be advantageous.

SUMMARY OF THE INVENTION

Thus, an object of the present invention relates to a production andpurification process for recombinant alpha-mannosidase.

In particular, it is an object of the present invention to provide ascalable production and purification process that solves the abovementioned problems of the prior art with providing sufficient amounts ofhigh purity alpha-mannosidase with high enzymatic activity therebyproviding a treatment for patients suffering from alpha-mannosidosis.Thus, one aspect of the invention relates to a process for purificationof recombinant alpha-mannosidase from a cell culture, wherein a fractionof said cell culture comprising recombinant alpha-mannosidase issubjected to chromatography on a resin comprising a multi-modal ligand.The inventors surprisingly found that this purification process resultedin composition comprising recombinant alpha-mannosidase with higherpurity and a higher percentage of the desired 130 kDa glycoproteinspecies than previously achieved. Achieving persistent high percentages(such as more than 80%) of the non-fragmented 130 kDa glycoprotein afterpurification is advantageous as this provides for a more uniform productas compared to a fragmented enzyme, which in turn enhances the abilityto obtain a pharmaceutical grade product.

Another aspect of the present invention relates to a process for fedbatch or continuous production of recombinant alpha-mannosidase,comprising the following steps:

a. inoculating a production reactor comprising a base medium with cellscapable of producing recombinant alpha-mannosidase on day 0, to providea cell culture;

b. adding a feed medium to said cell culture at least once from day 1;

c. adjusting the temperature of said cell culture to at the most 35° C.,such as 34° C., 33° C., 32° C., preferably at the most 31° C., eitherafter day 3 or when the viable cell density is higher than 2.1 MVC/mL,whichever comes first.

The inventors surprisingly found that the above production processresulted in a cell culture comprising recombinant alpha-mannosidase inhigh yields which was readily transferable to the purification column ofthe present invention without any dilution.

Yet another aspect of the present invention is to provide a compositioncomprising purified recombinant alpha-mannosidase, wherein at least 80%of the alpha-mannosidase is present as a 130 kDa glycoprotein.

One other aspect of the present invention is a composition comprisingpurified recombinant alpha-mannosidase for use in the treatment ofalpha-mannosidosis.

Yet another aspect of the present invention is a method of treatingalpha-mannosidosis and/or reducing or alleviating the symptomsassociated with alpha-mannosidosis, said method comprising a step ofadministering a composition comprising purified recombinantalpha-mannosidase to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an outline of the currently preferred purification processdesign for alpha-mannosidase from harvest to drug substance filing.

FIG. 2 shows an example of a Capto™ MMC column chromatogram foralpha-mannosidase.

FIG. 3 shows an example of a butyl Sepharose™ FF column chromatogram foralpha-mannosidase.

FIG. 4 shows an example of a CHT type 1 column chromatogram foralpha-mannosidase.

FIG. 5 shows an example of a Q Sepharose™ HP column chromatogram foralpha-mannosidase.

FIG. 6 shows an SDS-page chromatogram of the purified alpha-mannosidasecomposition indicating the distribution of the 130 kDa, 75 kDa and 55kDa glycoprotein species.

FIG. 7A shows an HPLC diagram for purified alpha-mannosidase using a 2step process where the amount of 130 kDa species is depicted as comparedto the 55 and 75 kDa species. The first peak from the left is the 55 kDaspecies, followed by the 130 kDa and 75 kDa species respectively. The 2step process is without the use of a multimodal ligand chromatographystep.

FIG. 7B shows an HPLC diagram for purified alpha-mannosidase using a 3step process where the amount of 130 kDa species is depicted as comparedto the 55 and 75 kDa species. The first peak from the left is the 55 kDaspecies, followed by the 130 kDa and 75 kDa species respectively. The 3step process uses a multimodal ligand chromatography step.

FIG. 7C shows an HPLC diagram for purified alpha-mannosidase using a 4step process where the amount of 130 kDa species is depicted as comparedto the 55 and 75 kDa species. The first peak from the left is the 55 kDaspecies, followed by the 130 kDa and 75 kDa species respectively. The 4step process uses a multimodal ligand chromatography step.

The present invention will now be described in more detail in thefollowing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

Prior to discussing the present invention in further details, thefollowing terms and conventions will first be defined:

Recombinant Alpha-Mannosidase

In the context of the present invention recombinant alpha-mannosidase isdefined as alpha-mannosidase which by virtue of its origin ormanipulation is not equal to all or a portion of the wild-typealpha-mannosidases found in nature. Thus, it is constructed usingrecombinant techniques which involves recombinant DNA molecules, that ishybrid DNA sequences comprising at least two fused DNA sequences, thefirst sequence not normally being fused with the second sequence innature. The recombinant alpha-mannosidase protein may be of human ornon-human origin. In particular, it may be a recombinant human lysosomalalpha-mannosidase (rhLAMAN). The alpha-mannosidase product may be asingle polypeptide or a mixture of a single polypeptide and fractionsthereof. Also the alpha-mannosidase may be subject to posttranslationalmodifications and may therefore be in the form of a glycoprotein.

Cell Culture

A cell culture is the process by which cells are grown under controlledconditions. In the present context the cells of the cell culture arespecifically designed to express a protein of interest, such asrecombinant alpha-mannosidase. The cell culture may reside in abioreactor, which is specially designed so as to allow control of thechemical and physical conditions.

Fraction

In the present context a fraction refers to a fraction of a cellculture. The fraction may constitute the whole cell culture, but isoften a treated fraction of the culture, such as a clarified, filtered,concentrated, diluted or partly purified fraction.

Resin

In the context of the present invention a resin constitutes the basis ofa stationary phase in a chromatography system, on which various chemicalgroups or substances are attached to provide a certain amount ofaffinity for a given molecule or protein of interest. Resins are oftenpolymeric beads with ligands covalently attached, said resins beinginsoluble in the liquid mobile phases used.

Multi-Modal Ligand

By multi-modal ligand is meant any ligand which is designed to interactwith a molecule or protein of interest in at least 2 ways. Theindividual interactions may independently be hydrophobic, hydrophilic,ionic, Van der Waals interactions, hydrogen bonding or any otherintermolecular chemical or physical interaction. In the present contexta ligand is an organic chemical substance attached to a resin as definedabove. A multi-modal ligand will have different affinities for differentsubstances that are passed through the chromatography column dissolvedin a mobile phase. The differences in affinity leads to variations inretention time of the different substances on the chromatography column,enabling separation of the substances. The retention times are alsodependent on other factors such as for example the constituents of themobile phase, pH and temperature. Resins comprising multimodal ligandsare sometimes referred to as “mixed mode” resins as well, but in thepresent context resins comprising a multi-modal ligand are not to beconfused with so-called “mixed-mode ion exchange resins” which compriseseveral different “ligands” on the same resin which may have oppositecharges, such as e.g. —OH, —Ca⁺ and —PO₄ ²⁻ in the case of ceramichydroxyapatite resin (CHT). In these resins the individual ligands arenot multi-modal.

Loading

In the present context loading refers to the transfer of a harvest,eluate or other solution onto a chromatographic system, such as achromatography column comprising a resin as a stationary phase.

Buffer

The term buffer is well known as a general description of a solutioncontaining either a weak acid and/or its corresponding salt or a weakbase and/or its corresponding salt, which is resistant to changes in pH.In the context of the present invention the buffers used are suitablefor use in chromatographic systems, such buffers include but are notlimited to: Phosphate buffers, e.g. disodium phosphate (Na₂HPO₄), sodiumphosphate or potassium phosphate, acetate buffers, e.g. sodium acetateor potassium acetate, sulphate buffers, e.g. sodium sulphate orpotassium sulphate, ammonium sulphate or Hepes, or other buffers, e.g.sodium borate or tris-HCl buffer.

Ultrafiltration

Ultrafiltration is a separation method in which hydraulic pressure isused to force molecules and solvent across a membrane comprising poresof a particular size, also known as the cut-off size or value. Onlymolecules which have a molecular weight smaller than the cut-off valueof the membrane are able to cross the membrane while those with a largermolecular weight do not cross the membrane and form the so calledretentate. The molecules present in the retentate may thereby beconcentrated as the solvent flows across the membrane.

In a particular embodiment the concentration of a solution orcomposition comprising a polypeptide such alpha-mannosidase may beperformed by Tangential flow filtration (TFF). This method is inparticular useful for large-scale concentration, i.e. for concentrationof solutions with a volume from one litre to several hundreds of litres.Thus this method is in particular useful for production of concentratedsolutions of a polypeptide of interests on an industrial scale. The TFFtechnique is based on the use of a particular apparatus which causes thesolution which is to be filtrated to flow across a semi-permeablemembrane; only molecules which are smaller than the membrane pores willpass through the membrane, forming the filtrate, leaving larger matterto be collected (retentate). With the TFF method two different pressuresare applied; one to pump the solution into the system and to circulateit in the system (inlet pressure), and another pressure is applied overthe membrane (membrane pressure) to force the small molecules and thesolvent across the membrane. The inlet pressure may typically be in therange of 1-3 bar, such as between 1.5-2 bar. The transmembrane pressure(TMP) may typically be larger than 1 bar. The concentrated compositionof a polypeptide of interest may be collected as the retentate when TFFis used to concentrate the composition. Membranes useful for TFF maytypically be made of regenerated cellulose or polyethersulphone (PES).

Diafiltration

In the present context diafiltration is a filtration process where aspecies of interest is in the retentate, i.e. it is not allowed to passthrough the filter, whereas other components such as for example buffersand salts do pass through the filter. Thus diafiltration may for examplebe used to exchange one buffer by another or to concentrate solutionscontaining a species of interest such as recombinant alpha-mannosidase.A first aspect of the present invention is to provide a process forpurification of recombinant alpha-mannosidase from a cell culture,wherein a fraction of said cell culture comprising recombinantalpha-mannosidase is subjected to chromatography on a resin comprising amulti-modal ligand. The advantage of using resins comprising amultimodal ligand in the present context is that these resins enablebinding of the alpha-mannosidase species in solutions having highconductivity levels. This has the advantage that undiluted harvest withhigh conductivity levels can be used, and no exchange of the harvestbuffer is necessary. The chromatography step comprising a multi-modalligand may therefore preferably be the first chromatography step afterisolating the fraction from the cell culture. Said chromatography stepcomprising a multi-modal ligand may often be referred to as a “capturestep”, since the protein of interest is initially withheld on the column(i.e. captured), while many impurities pass through the column duringwashing steps. The protein is subsequently eluted using a specificelution buffer.

Thus, in one embodiment of the invention a process is provided whereinthe fraction of the cell culture comprising the recombinantalpha-mannosidase is a clarified undiluted harvest. In the context ofthe present invention the term “clarified undiluted harvest” means aharvest of a cell culture, that is free of non-dissolved material orsolids, i.e. it is a clear solution. The harvest may have been submittedto a treatment in order to convert it to a clear solution. Suchtreatments may include but are not limited to: Filtration andcentrifugation. Furthermore, the harvest is not significantly dilutedprior to subjection to chromatography steps. Hence the harvest isdiluted by less than 10%, such as less than 7%, less than 5%, less than2%, less than 1%, less than 0.5%, such as less than 0.1%. In the mostpreferred embodiment the harvest is not diluted.

In another embodiment a process is provided wherein the clarifiedundiluted harvest has a conductivity of 10-20 mS/cm, such as 12-17mS/cm, preferably 15 mS/cm. The conductivity is measured prior toloading the harvest onto a chromatography system. In one embodiment saidchromatography is performed on a resin comprising a multimodal ligandhaving a carboxylic acid or sulphonic acid group. The carboxylic and/orsulphonic acids comprised in these ligands may be in the protonated formor in a deprotonated (salt) form depending on the conditions in thechromatographic system, particularly the pH of the mobile phase.

In yet another embodiment a process is provided wherein the resin boundmulti-modal ligand is a substance of formula (I), (II) or (III):

wherein R of the substances of formula (II) and (III) is a functionalgroup of formula (IV):

The multimodal ligand represented by the functional group of formula(IV) is generally referred to as “Cibracon Blue 3G” and examples ofcommercial products represented by the substances of formula (I), (II)and (III) are “Capto™ MMC”, “Capto™ Blue” and “Blue Sepharose™ fastflow” respectively. Other useful resins of the multimodal type include:Capto™ Adhere, MEP HyperCel™, HEA HyperCel™ and PPA HyperCel™ In thecontext of the present invention such resins have been proven especiallyeffective in the initial purification of an undiluted harvest comprisingrecombinant alpha-mannosidase.

An additional embodiment of the invention provides a process wherein thefraction of said cell culture loaded onto the resin comprising amultimodal ligand, is subjected to at least one washing step with asolution comprising isopropanol, preferably at least 1% (V:V)isopropanol, such as at least 2%, 3%, 4%, 4.5% (V:V) isopropanol,preferably at least 5% (V:V) isopropanol. The advantage of using asolution comprising isopropanol is that it provides for a better removalof unwanted host cell proteins (HCP's), specifically it helps to removea protease responsible for the proteolytic degradation of the desired130 kDa rhLAMAN species. HCP's are to understood as proteins endogenousto the host cell used in the cell culture during production. Althoughisopropanol is preferred other useful alcohols for this process includesethanol, n-propanol and n-butanol.

In yet another embodiment a process is provided wherein the pH of thesolution used for the washing step is in the range of pH 3.5-6.5, suchas pH 4.0-6.0, pH 4.5-5.5, preferably pH 4.7-5.0. Another embodimentprovides a process wherein the solution used for the washing stepcomprises an acetate buffer, preferably in a concentration in the rangeof 0.05-1.6 M, such as 0.1-1.5 M, 0.5-1.4 M, 0.7-1.3 M, 0.8-1.2 M,0.9-1.1 M, preferably 0.95 M. The acetate buffer may preferably beselected from the group consisting of sodium acetate, potassium acetate,lithium acetate, ammonium acetate.

Yet another embodiment provides a process wherein a first eluatecomprising recombinant alpha-mannosidase is eluted from the resincomprising a multi-modal ligand using an aqueous solution comprisingethylene glycol or propylene glycol. The addition of ethylene glycol tothe elution buffer was found to significantly enhance the yield ofeluted recombinant alpha-mannosidase. Propylene glycol was also enhancedyield but ethylene glycol is preferred.

One embodiment provides a process wherein the concentration of ethyleneglycol or propylene glycol in the aqueous solution is 20-60%, 20-50%,25-50%, 30-50%, 35-45%, such as 40%.

In a preferred embodiment a process is provided wherein the aqueoussolution comprising ethylene glycol or propylene glycol comprises sodiumchloride. The addition of sodium chloride to this solution was found tosignificantly enhance yields by promoting the elution of the rhLAMANenzyme.

In another embodiment the concentration of sodium chloride in theaqueous solution comprising ethylene glycol or propylene glycol is inthe range of 0.2 to 2.4 M, such as in the range of 0.4 to 2.2 M, 0.6 to2.0 M, 0.8 to 1.9 M, 1.0 to 1.8 M, 1.2 to 1.7 M, 1.4 to 1.6 M,preferably 1.5 M. Alternatively the concentration of sodium chloridemaybe in the range of 0.2 to 1.6 M or in the range of 1.4 to 2.4 M.

In a preferred embodiment the aqueous solution comprising ethyleneglycol or propylene glycol comprises a buffer. Said buffer maypreferably be a phosphate buffer, such as sodium phosphate or potassiumphosphate. Although phosphate buffers are preferred, additional usefulbuffers for the aqueous solution include citrate and borate buffers,Tris, MES, MOPS and Hepes buffers.

In another preferred embodiment the concentration of the buffering saltsin the aqueous solution comprising ethylene glycol or propylene glycolis 50-350 mM, 55-300 mM, 65-280 mM, 70-250 mM, 75-200, 80-200 mM, 85-150mM, preferably 90 mM.

In yet another preferred embodiment the pH of the aqueous solutioncomprising ethylene glycol or propylene glycol is pH 7.0-9.0, such as pH7.1-8.5, pH 7.2-8.3, pH 7.5-8.0, preferably pH 7.7.

In one embodiment a process is provided wherein a first eluatecomprising alpha mannosidase obtained from the resin comprising amulti-modal ligand is further subjected to a process comprising thesteps of

i) applying a fraction comprising alpha-mannosidase to a hydrophobicinteraction chromatography resin to provide an eluate comprising therecombinant alpha-mannosidase,

ii) passing a fraction comprising alpha-mannosidase through a mixed-modeion exchange resin to allow retention of contaminates to provide a flowthrough comprising the recombinant alpha-mannosidase, and

iii) subjecting a fraction comprising alpha-mannosidase tochromatography on a anion exchange resin to provide a eluate comprisingthe recombinant alpha-mannosidase.

In one embodiment a process is provided involving steps i)-iii) asdescribed above, wherein the fraction in step i) has been subject to apurification on said resin comprising a multimodal ligand, the fractionof step ii) is derived from the eluate from step i) and the fraction ofstep iii) is derived from the flow through from step ii). In other wordssteps i) to iii) are performed in the order they are listed, howeverwithout precluding intermediate steps in between steps i) to iii). Thesemay be intermediate purification steps and/or virus reduction or virusremoval steps. In a preferred embodiment the hydrophobic interactionchromatography resin of step i) is an alkyl substituted resin,preferably butyl sepharose resin. Alkyl substituted resins may includeethyl-, butyl- and octyl sepharose resins. Furthermore, phenyl sepharoseresins are also applicable. Examples of such resins are Butyl-SSepharose™ 6 Fast Flow, Butyl Sepharose™ 4 Fast Flow, Octyl Sepharose™ 4Fast Flow, Phenyl Sepharose™ 6 Fast Flow (high sub) and PhenylSepharose™ 6 Fast Flow (low sub), Butyl Sepharose™ High Performance,Phenyl Sepharose™ High Performance. The advantage of a purification stepinvolving hydrophobically interacting resins and particularly butylsepharose resin is effective removal of host cell proteins and DNAresidues, while retaining good yield of the rhLAMAN enzyme.

In yet another embodiment step i) comprises at least one washing step,wherein the solution used for washing comprises a phosphate buffer andan acetate buffer, preferably sodium phosphate and sodium acetate. Thisdual buffer washing step has proved especially effective in removingimpurities such as host cell proteins and DNA residues.

In yet another embodiment the concentration of phosphate buffer in thedual buffer washing of step i) is in the range of 5-40 mM, such as 10-30mM, 15-25 mM, preferably 20 mM, and the concentration of acetate bufferis in the range of 0.9-1.5 M, such as 1.0-1.4 M, 1.1-1.3 M, preferably1.2 M.

In another embodiment step i) comprises at least one washing step,wherein the solution used for washing comprises no more than one buffer,preferably a phosphate buffer, preferably sodium phosphate.

In another embodiment the one buffer of the at least one washing stepcomprising no more than one buffer is present in a concentration in therange of 0.4-0.8 M, such as 0.5-0-7 M, preferably 0.6 M.

In one embodiment a process is provided wherein the mixed-mode ionexchange resin of step ii) is a ceramic hydroxyapatite or fluoroapatiteresin, preferably ceramic hydroxyapatite type I (CHT I) resin. Applyingthis chromatography step has been shown to efficiently separate asignificant amount of DNA impurities from the recombinantalpha-mannosidase composition and bind host cell proteins while therhLAMAN enzyme product passes the column without binding.

In another embodiment the anion exchange resin of step iii) is a stronganion exchange resin, such as a quaternary ammonium strong anionexchange resin. Such resins are included but not restricted to thefollowing examples: Q-Sepharose™ HP, Q-Sepharose™ FF, DEAE-Sepharose™,Capto™ Q, Uno™ Q, ANX Sepharose™.

In yet another embodiment a process is provided wherein a virusinactivation step is performed, preferably in between step ii) and stepiii). In a preferred embodiment the virus inactivation step comprisesmixing the flow through of step ii) with an aqueous solution ofisopropanol (1:1 V/V of flow through/aqueous isopropanol) for at least 2hours, preferably followed by concentration by ultrafiltration andremoval of isopropanol using diafiltration. The aqueous isopropanolduring inactivation may be in the range of 10-50% isopropanol, such as20-40%, 25-35%, 28-32%, preferably 30% isopropanol. The 1:1 V/V solutionof flow through and aqueous isopropanol thus has a final concentrationof isopropanol of 15%.

Another preferred embodiment is a process wherein a virus reduction stepis performed, preferably after chromatography step iii).

In one embodiment the virus reduction step comprises filtration of asolution comprising recombinant alpha-mannosidase, preferably the eluateof step iii), through a filter, preferably a virus removal filter, suchas a Ultipor™ VF grade DV20 filter, or a Planova™ 15N or 20N filter,Preferably a Planova™ 15N filter is used.

The purification process of the present invention may advantageously beperformed on a large scale, thus in preferred embodiments the process isperformed on chromatography columns having a column volume of at least0.5 L, such as at least 1.0 L, 2.0 L, 5.0 L, 10 L, preferably at least13.0 L.

In another embodiment of the present invention a purification process asdescribed above is provided, wherein the alpha-mannosidase has asequence selected from:

A) the sequence set forth in SEQ ID NO 2

B) an analogue of the sequence in A

C) a subsequence of the sequence in A) or B)

Where the sequence described by SEQ ID NO 2 represents the amino acidsequence for the recombinant human lysosomal alpha-mannosidase (rhLAMAN)as provided in WO 02/099092.

By “subsequence” is meant a fragment of the parent sequence having asize of no less than 50% of the parent sequence, such as no less than55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or no less 95% of the parentsequence. Accordingly, the subsequences in question may have a length offrom 505-1009 consecutive amino acid residues, such as from 525-1009,from 550-1009, 575-1009, 600-1009, 625-1009, 650-1009, 675-1009,700-1009, 725-1009, 750-1009, 775-1009, 800-1009, 825-1009, 850-1009,875-1009, 900-1009, 925-1009, 950-1009, 975-1009, 980-1009, 990-1009 orsuch as from 1000-1009 consecutive amino acid residues. Furthermore, therelevant subsequences of SEQ ID NO: 2 or analogues thereof must retainthe catalytic site. Although the 3D structure of human LAMAN is unknown,the 3D structure of the bovine LAMAN has been reported and based on thatdata it has been concluded that the following amino acids participate inthe active site and/or are responsible for coordinating the Zn²⁺ atomrequired for activity also in human LAMAN: AA 72=H, AA 74=D, AA 196=D,AA 446=H (UniProtKB/Swiss-Prot database: 000754, MA2B 1_HUMAN_,Heikinheimo et al. J. Mol. Biol. 327, 631-644, 2003). It has been shownthat mutations of AA 72 and 196 in human LAMAN results in almostcomplete loss of enzyme activity (Hansen et al., Biochem. J. (2004),381, pp. 537-567). In order to display activity, a subsequence of therhLAMAN should retain at least the regions containing the above fouramino acids. Preferably, the subsequences of rhLAMAN also comprise oneor more additional conformational parts, including for example bindingsites, beta-turns, disulfide bridges, stop codons and others. In thehuman form of LAMAN there are several disease causing mutationsindicating importance for that particular amino acid, e.g. AA 53, 72,77, 188, 200, 355, 356, 359, 402, 453, 461, 518, 563, 639, 714, 750,760, 801, 809, 916 (The human gene mutation database, HMDG®professional, Cardiff University, 2009) and there are also amino acidswhich are of importance for glycosylations, including AA 133, 310, 367,497, 645, 651, 692, 766, 832, 930 and 989 and amino acids involved indisulfide bridges such as AA 55+358, 268+273, 412+472 and 493+501.

By “analogue” is meant a sequence with a certain percentage of sequenceidentity with the parent sequence, this may be at least 60% sequenceidentity, such as at least 70%, 80%, 85%, 90%, 95%, 98% or preferably99% sequence identity It will be understood that the analogues andsub-sequences set forth above are preferably functionally equivalent tothe alpha-mannosidase having the amino acid sequence set forth in SEQ IDNO: 2 in the sense that they are capable of exerting substantially thesame enzymatic activity.

The term “substantially the same enzymatic activity” refers to anequivalent part or analogue having at least 50%, preferably at least60%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95% and most preferably at least97%, at least 98% or at least 99% of the activity of the natural enzyme.An example of a functionally equivalent analogue of the enzyme could bea fusion protein which includes the catalytic site of the enzyme in afunctional form, but it can also be a homologous variant of the enzymederived from another species. Also, completely synthetic molecules thatmimic the specific enzymatic activity of the relevant enzyme wouldconstitute “functionally equivalent analogues”. Non-human analogues ofLAMAN are generally not applicable for therapy as they can potentiallyinduce the formation of antibodies in the patient and cause disease.Human analogues however may be useful in enzyme replacement therapy,when the mutations are not disease causing and do not diminish thedesired enzyme activity significantly. Examples of such mutations are:His70Leu, Gln240Arg, Ser250Ala, Leu278Val, Ser282Pro, Thr312Ile,Ala337Arg, Ser413Asn, Ser481Ala, Gln582Glu, Arg741Gly, Thr873Pro(Source: www.ensembl.org.; transcript ID ENST00000456935). AlsoPro669Leu and Asp402Lys are known by the inventors not to cause disease.Generally, the skilled person will be able to readily devise appropriateassays for the determination of enzymatic activity. For LAMAN, anappropriate enzyme activity assay is disclosed in WO 02/099092 page 26,lines 8-28. Briefly, the following procedure may be performed forscreening purposes using flat-bottomed 96-well plates: 75 μl of 4× assaybuffer (8 mM p-Nitrophenyl-alpha-D-mannopyranoside, 2 mg/mL BSA, 0.4 MNa Acetate (pH 4.5) is added to 75 μl of sample or an appropriatedilution of it (in 10 mM Tris pH 7.4 containing 150 mM NaCl+10%superblock). The plates are incubated at 37 deg C. for 30 min andstopped with 75 μl of 1.8 M Na₂CO₃ and the absorbance recorded at 405 nmon a plate reader. The 96-well plates are read on a spectrophotometer.Specific activity is defined as μmoles ofp-Nitrophenyl-alpha-D-mannopyranoside hydrolysed per minute per mgprotein.

As commonly defined “identity” is here defined as sequence identitybetween genes or proteins at the nucleotide or amino acid level,respectively. Thus, in the present context “sequence identity” is ameasure of identity between proteins at the amino acid level and ameasure of identity between nucleic acids at nucleotide level. Theprotein sequence identity may be determined by comparing the amino acidsequence in a given position in each sequence when the sequences arealigned. Similarly, the nucleic acid sequence identity may be determinedby comparing the nucleotide sequence in a given position in eachsequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps may be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. One may manually align the sequences and count the number ofidentical amino acids. Alternatively, alignment of two sequences for thedetermination of percent identity may be accomplished using amathematical algorithm. Such an algorithm is incorporated into theNBLAST and XBLAST programs. BLAST nucleotide searches may be performedwith the NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleic acid molecules of the invention. BLASTprotein searches may be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may beused to perform an iterated search, which detects distant relationshipsbetween molecules. When utilising the NBLAST, XBLAST, and Gapped BLASTprograms, the default parameters of the respective programs may be used.See www.ncbi.nlm.nih.gov. Alternatively, sequence identity may becalculated after the sequences have been aligned e.g. by the BLASTprogram in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).Generally, the default settings with respect to e.g. “scoring matrix”and “gap penalty” may be used for alignment. In the context of thepresent invention, the BLASTN and PSI BLAST default settings may beadvantageous.

The percent identity between two sequences may be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

Another embodiment of the present invention is a composition comprisingalpha-mannosidase obtainable by the purification process describedabove. In a second aspect of the present invention a process is providedfor fed batch or continuous production of recombinant alpha-mannosidase,comprising the following steps: a. inoculating a production reactorcomprising a base medium with cells capable of producing recombinantalpha-mannosidase on day 0, to provide a cell culture; b. adding a feedmedium to said cell culture at least once from day 1; c. adjusting thetemperature of said cell culture to at the most 35° C., such as 34° C.,33° C., 32° C., preferably at the most 31° C., either after day 3 orwhen the viable cell density is higher than 2.1 MVC/mL, whichever comesfirst.

In the above process inoculation day is defined as day 0, and thefollowing day is day 1 and so on. The starting temperature used from day0 until the adjustment described in point c. is in the range 36-37° C.,preferably 36.5° C. It is to be understood that the abovementionedtemperatures are actual measured temperatures, not set points, i.e. inthe bioreactor setup used for the present invention the temperature of31° C. mentioned above required a temperature set point of 32° C.Likewise a temperature of 36.5° C. requires a set point temperature of37° C.

Suitable host cells for the expression and production of recombinantalpha-mannosidase are derived from multicellular organisms, preferablyfrom mammals.

In particular, the cells used to produce recombinant alpha-mannosidasemay be selected from the group consisting of monkey kidney CVI linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture); baby hamster kidneycells (BHK); Chinese hamster ovary cells/-DHFR (CHO); mouse Sertolicells (TM4); monkey kidney cells (CV I); African green monkey kidneycells (VERO-76); human cervical carcinoma cells (HELA); canine kidneycells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138);human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562);TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).Insect cell lines or human fibroblast cells are also available assuitable host cells. Production of recombinant alpha-mannosidase isobtained using cells transfected with an appropriate nucleic acidconstruct using techniques known to the skilled person. In particular,the nucleic acid construct may comprise a nucleic acid sequence selectedfrom the group consisting of: i) the nucleic acid sequence set forth inSEQ ID NO: 1; and ii) a nucleic acid sequence coding for a sub-sequenceor analogue of the sequence set forth in SEQ ID NO 2 as provided above.

The cells may preferably be a rLAMAN Chinese Hamster Ovary (CHO) cellline developed specifically for the purpose of producing recombinantenzyme as described in WO 02/099092. A culture of this cell line DSMACC2549 which was deposited at the DSMZ GmbH, Maschroderweg 1b, D-38124Braunschweig, Germany for the purpose of patent deposit according to theBudapest treaty on 6 Jun. 2002. This cell may be obtained using theexpression plasmid pLamanExpi having the sequence shown in SEQ ID NO 1.

The process of steps a-c may further comprise the following step: d. Aprocess for purification of recombinant alpha-mannosidase from said cellculture, wherein a fraction of said cell culture comprising recombinantalpha-mannosidase is subjected to chromatography on a resin comprising amulti-modal ligand having a carboxylic acid or sulphonic acid group, asdescribed above.

In yet another embodiment the cell culture used in the productionprocess is essentially free of any supplements derived from animals,such as cod liver oil supplements. Avoiding the use of such supplementsreduces the risk of viral contamination in the final enzyme product.

In one preferred embodiment of the production process the undilutedharvest of the fed batch or continuous production has a concentration ofalpha-mannosidase of at least 0.1 g/L, such as at least 0.2 g/L, 0.3g/L, 0.4 g/L, preferably at least 0.5 g/L.

In another embodiment the undiluted harvest of the fed batch orcontinuous production has an enzyme activity in the range of 3-35 U/mL,such as 5-35 U/mL, 7-35 U/mL, preferably in the range of 10-35 U/mL. Itis to be understood that upon further process optimization the enzymeactivity of the harvest may become even higher than 35 U/mL.

The production process may advantageously be performed at a large scale.Thus in one embodiment the process for fed batch or continuousproduction is performed at a volume of at least 30 L, such as at least50 L, 75 L, 100 L, 150 L, 200 L, preferably at least 250 L.

In another embodiment of the present invention a production process asdescribed above is provided, wherein the alpha-mannosidase has asequence selected from:

A) the sequence set forth in SEQ ID NO 2

B) an analogue of the sequence in A

C) a subsequence of the sequence in A) or B)

Another embodiment of the present invention is a composition comprisingalpha-mannosidase obtainable by the production process described above.Such compositions may in preferred embodiments comprise additionalactive product ingredients (API), adjuvants, and/or excipients.

In a third aspect of the present invention a composition is providedcomprising purified recombinant alpha-mannosidase, wherein at least 80%of the alpha-mannosidase is present as a 130 kDa glycoprotein.

In a preferred embodiment the composition comprising purifiedrecombinant alpha-mannosidase is provided wherein the recombinantalpha-mannosidase remains stable in liquid solution for at least 4 dayswhen stored at +5° C. or for at least 24 months when stored at −20° C.

The currently preferred composition for the formulation buffer solutionfor the rhLAMAN enzyme product is described below and the achievedstabilities are also listed:

Na₂HPO₄ 3.50 mM (Dibasic sodium phosphate)

NaH₂PO₄ 0.17 mM (Monobasic sodium phosphate)

Glycine 27 mM

Mannitol 250 mM

pH 7.70, 290 mOsm/kg (isotonic solution)

In-use stability:

Stable solution  +5° C.-4 days +20° C.-6 hours −20° C.-24 months FreezeDried  +5° C.-24 months

In another embodiment of the present invention a composition asdescribed above is provided, wherein the alpha-mannosidase has asequence selected from:

A) the sequence set forth in SEQ ID NO 2

B) an analogue of the sequence in A

C) a subsequence of the sequence in A) or B)

Another preferred embodiment is the above composition comprisingpurified recombinant alpha-mannosidase for use as a medicament.

In a further embodiment the above composition comprising purifiedrecombinant alpha-mannosidase is for use in the treatment ofalpha-mannosidosis.

Yet another embodiment is the use of the above composition comprisingpurified recombinant alpha-mannosidase for the preparation of medicamentfor the treatment of alpha-mannosidosis. Another embodiment is a methodof treating alpha-mannosidosis and/or reducing or alleviating thesymptoms associated with alpha-mannosidosis, said method comprising astep of administering a composition comprising a purified recombinantalpha-mannosidase as provided above to a subject in need thereof.

It should be noted that embodiments and features described in thecontext of one of the aspects of the present invention also apply to theother aspects of the invention.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the followingnon-limiting examples.

EXAMPLES Abbreviations Used

CIP: Clean In Place

CV: Column Volume

Cv: viable cell density

DF: Diafiltration

DO: Dissolved Oxygen

IPA: Isopropanol

MVC/mL: 10⁶ viable cells/mL

NaPi: Sodium Phosphate

NaAc: Sodium Acetate

OD: Optical Density

EG: ethylene Glycol

TFF: Tangential Flow Filtration

TMP: Transmembrane pressure

UF: Ultrafiltration

Example 1 Currently Preferred Overall Purification Procedure

The purification procedure for obtaining the optimum yields and puritiesfor alpha-mannosidase in the context of the present invention is asdescribed below. Standard conditions for regeneration and cleaning ofresins are used, as prescribed for the individual resin (see alsoexamples 2-5). The resins used are available at GE healthcare lifesciences and BioRad.

-   -   Providing fraction of a harvest from a production of        alpha-mannosidase, and clarifying the fraction without        significant dilution. Preferably no dilution at all is made.    -   Performing a capture step involving column chromatography of the        above fraction on a resin comprising a multimodal ligand. These        resins are selected from the group consisting of: Capto™ MMC,        Capto™ Adhere, PlasmidSelect™ Xtra, Capto™ Blue, Blue Sepharose™        Fast Flow resin, MEP HyperCel™, HEA HyperCel™ and PPA HyperCel™.

Several washes are performed at pH 4.5-8.5, and the wash buffers areselected from the group consisting of: sodium acetate, potassiumacetate, ammonium acetate, sodium phosphate, potassium phosphate, sodiumsulphate, potassium sulphate, ammonium sulphate, MES, MOPS, Hepes,sodium borate, tris-HCl, citrate buffer or combinations thereof (buffergroup A, hereinafter). In at least one wash however, the washingsolution comprises isopropanol and the pH is between pH 4-6. The elutionbuffer is selected from buffer group A, and the elution solutioncomprises ethylene glycol. Elution pH is kept at pH 7.0-8.5.

-   -   Performing an active intermediate step involving column        chromatography of a composition comprising alpha-mannosidase on        a hydrophobic interaction resin. These resins are selected from        the group consisting of: Butyl-S Sepharose™ 6 Fast Flow, Butyl        Sepharose™ 4 Fast Flow, Octyl Sepharose™ 4 Fast Flow, Phenyl        Sepharose™ 6 Fast Flow (high sub) and Phenyl Sepharose™ 6 Fast        Flow (low sub), Butyl Sepharose™ High Performance, Phenyl        Sepharose™ High Performance.

Several washes are performed at pH 7-8. The wash buffers are selectedfrom buffer group A. The elution buffer is also selected from buffergroup A and elution pH is between pH 7-8.

-   -   Performing a passive intermediate step involving column        chromatography of a composition comprising alpha-mannosidase on        a mixed-mode ion exchange resin to provide a flow-through. These        resins are selected from the group consisting of: ceramic        hydroxyapatite type I or II (preferably type I) resin, or        fluoroapatite. One wash is performed to provide a flow-through        at pH 7-8. The wash buffer is selected from buffer group A.    -   Performing a polishing step involving column chromatography of a        composition comprising alpha-mannosidase on an anion exchange        resin. These resins are selected from the group consisting of:        Q-Sepharose™ HP, Q-Sepharose™ FF, DEAE-Sepharose™, Capto™ Q,        Uno™ Q, ANX Sepharose™.

Several washes are performed at pH 7-8. The wash buffers are selectedfrom buffer group A and TRIS-HCl buffer. The elution buffer is alsoselected from buffer group A and elution pH is kept between pH 7-8.

-   -   Performing a virus inactivation step by bringing a solution of        the alpha-mannosidase in contact with isopropanol.    -   Performing a virus removal step using nano-filtration.

The yields and product components ratios for the purified compositioncomprising alpha-mannosidase according to the present invention areshown in the below table 1, with comparison to previous methods,wherein:

Method 1 is: The currently preferred method according to the presentinvention.

Method 2 is: Similar to method 1 without a polishing step. It is alsowithout washing steps comprising isopropanol for the capture step andwith fewer washes in the active intermediate step, and finally it iswithout virus inactivation/removal steps.

Method 3 is: As described in WO 02/099092 (multimodal ligands not used).

TABLE 1 yields and purities resulting from past and current purificationprocedures Scale Overall Overall % (Culture Method yield purity % 130kDa % 55 kDa 75 kDa volume) 1 70% 99.6% 95.2% 1.5% 2.9% 250 L 2 60-70%98.2% 92.1% 2.6% 3.5%  30 L 3 70-80%   80%  <80%  >5%  >5%  1 L

See also example 10 and FIG. 7.

Example 2 Chromatographic Capture Step Using a Multi Modal Ligand

Clarified undiluted harvest comprising alpha-mannosidase binds by mixedmode interaction to a multimodal ligand type resin such as Capto™ MMC asused in this example. Increasing salt and addition of ethylene glycolelutes the product. The capacity of Capto™ MMC was 260 U/ml resin. Thecapture stage was performed using the following steps:

-   -   Regenerate the column with 1-2 column volumes (CV) of 3 M NaCl,        pH 10-12 at 300 cm/hr.    -   Equilibrate with 5 CV of 50 mM sodium phosphate buffer (NaPi),        0.15 M NaCl pH 7.5 at 300 cm/hr.    -   Load clarified, undiluted harvest (conductivity ˜15 mS/cm) at        300 cm/hr.    -   Wash 1: 4 CV of equilibration buffer.    -   Wash 2: 3 CV of 0.95 M NaAc, 5% (v:v) isopropanol, pH 4.9 at 300        cm/hr.    -   Wash 3: 4 CV of equilibration buffer (until stable baseline,        ˜0.06 Au with 5 mm flowcell) of equilibration buffer at 300        cm/hr.    -   Elute the product with 6 CV of 1.5 M NaCl, 40% ethylene glycol        in 90 mM NaPi, pH 7.7 at maximum 120 cm/hr. Start collecting        when the absorbance increases (around 10 mAu from the new        baseline). Collect ˜4 CV.    -   Regenerate the column, as above with 3 CV, downward flow        direction, maximum 120 cm/hr.    -   Clean in place (CIP) and sanitize, preferably with upward flow        direction, with 3 CV H₂O, 3 CV 1 M NaOH (˜60 minutes contact        time), 2-3 CV of phosphate buffer, pH ˜7, 3 CV 20 mM sodium        phosphate+20% ethanol. Store in 20 mM sodium phosphate+20%        ethanol.

Table 2 shows an example of a purification scheme. Table 3 summarizesthe step. FIG. 2 shows an example of a chromatogram for this step.

TABLE 2 Capture stage purification scheme: Capto ™ MMC packed in a 13.5× 2 ml (27 ml) XK 16 column OD 280 Vol. Activity Total divided Yield HCPStep (ml) (U/ml) activity (U) by 1.8 (%) μg/ml Load 439 15 6585 (=244U/ml resin) Flow 560 ~37 0.6 through + 1^(st) equilibration (eq) bufferwash 0.95M 90 0 NaAc + 5% IPA, pH 4.9 Eq. buffer 101 0.3 30 0.5 Eluate118 50 5900 2.2 89 28

TABLE 3 Summary of conditions for Capto ™ MMC capture stage Flow rateVolume Flow Step Buffer (cm/hr) (CV) Direction Regeneration 3M NaCl, pH~11 300 2 down Equilibration 50 mM NaPi, 150 mM 300 5 down NaCl, pH 7.5Load Harvest 300 down Wash 1 50 mM NaPi, 150 mM 300 4 down NaCl, pH 7.5Wash 2 0.95M NaAc, 5% IPA, 300 3 down pH 4.8 Wash 3 50 mM NaPi, 150 mM300 4 down NaCl, pH 7.5 Elution 90 mM NaPi, 1.5M NaCl, ≦120 6 down 40%EG, pH 7.7 Regeneration 2M NaCl, pH ~11 120 3 down Flush Water 300 3 upCIP 1M NaOH 300 3 up Conditioning Phosphate buffer (RB to 300 1-3 updecide) Storage 20 mM NaPi + 20% 100 3 up Ethanol

Example 3 Chromatographic Intermediate Active Step Using HydrophobicInteraction

The product from the capture stage comprising alpha-mannosidase binds byhydrophobic interactions after addition of sodium sulfate to hydrophobicinteraction type resins, such as Butyl Sepharose™ 4 FF as used in thisexample. Reducing the salt concentration elutes the product. Thecapacity was 195 U/ml resin. The following steps were used in theintermediate active stage:

-   -   Regenerate the column with 1 CV 20 mM sodium phosphate (NaPi)        buffer, pH 7.5 at 100 cm/hr.    -   Equilibrate the column with 5 CV 0.5 M Na₂SO₄, 20 mM NaPi, pH        7.5 at 150 cm/hr.    -   Mix the product pool from step 1 with the same volume 20 mM        NaPi, 0.8 M Na₂SO₄, pH 7.5 and load it onto the column at 70        cm/hr. The mixing can be performed in-line or maximum 3 hours        before loading starts. The 1:1 volume:volume (v:v) mix        corresponds to approximately 1.11:1, weight:weight (w:w)        (eluate:sodium sulfate buffer). If needed the conditioned load        should be filtered through 0.45 μm filter (hydrophilic PES or        PVDF) before loading.    -   Wash the column with 3 CV of equilibration buffer at 70 cm/hr to        remove ethylene glycol, in addition to host cell proteins, from        the previous step.    -   Wash with 3.5 CV of 20 mM NaPi, 1.2 M NaAc, pH 7.5 at 100 cm/hr.    -   Wash with 3.5 CV of 0.6 M NaPi, pH 7.0 at 150 cm/hr.    -   Elute the product with 4 CV of 60 mM NaPi, pH 7.5 at 150 cm/hr.        Collect the peak from the initial increase of absorbance until        baseline is reached, ˜2 CV.    -   Regenerate the column with 2 CV 20 mM NaPi, pH 7.5 followed by 3        CV H₂O at 150 cm/hr.    -   Clean and sanitize with 3 CV 1 M NaOH (60 min contact time), 1        CV H₂O, 1-3 CV of phosphate buffer and 2 CV 20 mM sodium        phosphate+20% ethanol. Store in 20 mM sodium phosphate+20%        ethanol.

Table 4 shows an example of a purification scheme. Table 5 summarizesthe step. FIG. 3 shows an example of a chromatogram for this step.

TABLE 4 Intermediate active purification scheme using Butyl Sepharose ™4FF packed in a 13.5 cm H 2 cm² (27 ml) XK 16 column OD 280 VolumeActivity Total divided Yield HCP Step (ml) (U/ml) activity (U) by 1.8(%) ng/mg Load 236 23 5413 (=200 U/ml resin) Flow ~390 0.3 ~100 1.8through + eq buffer wash 1.2M NaAc, 95 0.5 48 0.8 pH 7.5 0.6M NaPi, 970.2 19 0.3 pH 7.0 Eluate 66 81 5346 3 98 940

TABLE 5 Summary of conditions for Butyl Sepharose ™ 4FF step Flow rateVolume Flow Step Buffer (cm/hr) (CV) Direction Regeneration 20 mM NaPi,pH 7.5 150 1 down Equilibration 20 mM NaPi, 0.5M sodium 150 5 downsulfate, pH 7.5 Load Conditioned Capto MMC 70 ~6 down eluate Wash 1 20mM NaPi, 0.5M sodium 70 3 down sulfate, pH 7.5 Wash 2 20 mM NaPi, 1.2MNaAc, 100 3.5 down pH 7.5 Wash 3 0.6M NaPi, pH 7.0 150 3.5 down Elution60 mM NaPi pH 7.5 150 4 down Regeneration 20 mM NaPi, pH 7.5 150 2 downFlush water 150 3 up CIP 1M NaOH 150 3 up Flush water 150 1 upConditioning Phosphate buffer (RB to 150 1-3 up decide) Storage 20 mMNa-Pi + 20% 150 3 up Ethanol

Example 4 Chromatographic Intermediate Passive Step Using Mixed-Mode IonExchange

Two eluates comprising alpha-mannosidase from the intermediate activestep were pooled and mixed 1:1 (weight:weight) with water to reduce theconductivity and loaded onto a mixed-mode ion exchange resin, such as inthis example a Ceramic Hydroxyapatite I (CHT I) resin. The productpasses without binding, while host cell proteins bind to the column. Theflow through, containing the product was collected. The capacity was 550U/ml resin. The following steps were used in this example of anintermediate passive stage:

-   -   Regenerate the column with 2 CV of 0.6 M NaPi pH 7.0 at 300        cm/hr.    -   Equilibrate the column with 5 CV of 60 mM NaPi, pH 7.5 at 300        cm/hr.    -   Load the conditioned eluate from step 2 at 300 cm/hr and collect        the flow through, containing the product. The conductivity and        pH of the load will be ˜10 mS/cm and 7.3, respectively. Collect        the flow through, containing the product from an OD increase of        20 mAu until OD is back to 20 mAU, approximately the loading        volume and 2 CVs wash. End-of step filter the product pool        through 0.45 μm hydrophilic PES or PVDF filter.    -   Wash the column with 4 CV equilibration buffer at 300 cm/hr.    -   Regenerate the column with 3 CV of 0.6 M NaPi, pH 7.0 at 300        cm/hr.    -   Clean and sanitize with 3 CV 1 M NaOH (60 min contact time), 1        CV of 60 mM NaPi, pH 7.5 and 2 CV 20 mM sodium phosphate+20%        ethanol. Store in 20 mM sodium phosphate+20% ethanol.

Table 6 shows an example of a purification scheme. Table 7 summarizesthe step. FIG. 4 shows an example of a chromatogram for this step.

TABLE 6 Purification scheme for intermediate passive stage using CHT Ipacked in a 10 cm × 2 cm2 (20 ml) XK 16 column OD 280 Volume ActivityTotal divided Yield HCP Step (ml) (U/ml) activity (U) by 1.8 (%) ng/mgLoad 360 31.6 11390 (=570 U/ml resin) Flow 392 27.5 10780 95 ~500through = product end-of step filtered 0.5M NaPi 40 0.45   18

TABLE 7 Summary of conditions for CHT I step Flow rate Volume Flow StepBuffer (cm/hr) (CV) Direction Regeneration 600 mM NaPi, pH 7.0 300 2down Equilibration 60 mM NaPi, pH 7.5 300 5 down Load Butyl eluate + H₂O300 down Wash 1 60 mM NaPi, pH 7.5 300 4 down Regeneration 600 mM NaPi,pH 7.0 300 3 down CIP 1M NaOH ≦300 3 up Conditioning NaPi buffer 300 1up (RB to decide) Storage 20 mM NaPi + 20% 300 3 up Ethanol

Example 5 Virus Inactivation Step

Virus inactivation may be performed at different stages of the process.In this example the virus inactivation was performed after theintermediate passive step and prior to the polishing step. Virusinactivation of the intermediate passive pool comprisingalpha-mannosidase is obtained by 135±15 min incubation at 21±5° C. with15% isopropanol (1:1 mixture with 30% aqueous isopropanol). The tank canbe cooled by a cooling jacket at +4° C. to keep the process at 21±5° C.Tangential flow filtration (TFF), with 100 kDa polyethersulfonemembrane, Screen A (from Millipore or Sartorius) was used to remove theisopropanol and change to sodium phosphate buffer. The following stepswere used to inactivate viruses in this example:

-   -   Mix the product (flow through) from the intermediate passive        step with 30% isopropanol in 60 mM sodium phosphate, 1:1 (v:v),        which corresponds to 1:0:94 (w/w). Mix, e.g. by recirculation        pumping. The product protein concentration will be ˜0.3-1 mg/ml.    -   Incubate the solvent/product pool at room temperature for 135±15        min.    -   Equilibrate the TFF membrane with 60 mM sodium phosphate buffer.    -   Concentrate the pool to target concentration 2 mg/ml (0.5-3        mg/ml) by ultrafiltration at transmembrane pressure (TMP) 1.1        bar, at 21±5° C. pressure in =˜1.4-1.5 bar and pressure        out=0.7˜-0.8 bar.    -   Exchange ˜6 volumes by diafiltration against 60 mM sodium        phosphate buffer. Start at TMP 1 bar (1.4 bar in/0.6 bar out).        After the first volume is exchanged the TMP can be increased to        1.1.    -   Collect the retentate. Rinse the membrane with 2-3 system        volumes of dilution buffer to remove loosely bound product.        Collect the rinse together with the retentate. The final target        protein concentration is 2 mg/ml (0.5-3 mg/ml).    -   Clean the membrane with H₂O, followed by 0.5 M NaOH (60 min        contact time). Store in 0.1 M NaOH.

Table 10 shows the conditions for the virus inactivation/TFF step

TABLE 10 Summary of conditions for virus inactivation/TFF step Dil. TMPStep Buffer w:w UF DF bar Comment Start 30% IPA/ 1.94x 135 ± 15 min, RT70% NaPi Equilibration NaPi UF ~5x 1.1 Concentrate to 2 mg/ml (1.5in/0.7(0.5-3 mg/ml), flux out) ~100-65 LMH DF NaPi 6x 1.0-1.1 First volume atlower (1.5in/0.7 TMP (1.4in/0.6out), out) then increase to TMP 1.1, flux~65-100 LMN System wash NaPi 2 system volumes, pool with retentate totarget concentration 2 mg/ml (0.5-3 mg/ml) Rinse water CIP 0.5-1M NaOHstore 0.1M NaOH NaPi = 60 mM sodium phosphate, pH 7.5

Example 6 Chromatographic Polishing Step Using Anion Exchange

The conductivity of the retentate comprising alpha-mannosidase from theintermediate passive stage, was reduced by dilution 6 times withconditioning buffer (20 mM Tris-HCl, 10 mM NaCl, 75 mM mannitol, 0.005%Tween™ 80, pH 7.5) in order to bind by ionic interaction onto a anionexchange resin, such as in this example a quarternary ammonium highperformance strong anion exchange resin (Q Sepahrose™ HP resin). Theretentate was either diluted directly before loading or by in-linedilution. The product was eluted, into a container prefilled with 1 CVof elution buffer, by addition of sodium chloride. The capacity is 400U/ml resin. The following steps were used for the polishing stage inthis example:

-   -   Regenerate the column with 1 CV 50 mM NaPi, 1 M sodium chloride,        pH 7.5 at 120 cm/hr.    -   Equilibrate the column with 5 CV 20 mM Tris-HCl, 10 mM sodium        chloride, pH 7.5 at 120 cm/hr.    -   Load the diluted retentate from step 4 at 120 cm/hr.    -   Wash the column with 5 CV of equilibration buffer and 1 CV of 20        mM sodium phosphate, pH 7.5 at 120 cm/hr.    -   Elute the product with 4 CV of 50 mM sodium phosphate, 0.2 M        sodium chloride, pH 7.5 at 120 cm/hr into the prefilled (1 CV of        elution buffer) container. Collect the peak from the initial        increase (start collect at 10-20 mAu) of absorbance until 30-50        mAu (2 mm flowcell), 0.5-1.5 CV.    -   Regenerate the column with 3 CV 50 mM NaPi, 1 M sodium chloride,        pH 7.5 at 120 cm/hr.    -   Clean and sanitize with 3 CV 1 M NaOH (60 min contact time) and        3 CV 10 mM NaOH. Store in 10 mM NaOH.

Table 8 shows an example of a purification scheme. Table 9 summarizesthe step. FIG. 5 shows an example of a chromatogram.

TABLE 8 Example of purification scheme for polishing step using QSepharose ™ HP resin 19 cm × 2 cm² (38 ml) OD 280 Volume Activity Totaldivided Yield HCP Step (ml) (U/ml) activity (U) by 1.8 (%) ng/mg Load1095 9.6 10500 (=276 U/ml resin) Flow ~1300 0.07 ~90 ~1 through 20 mM 751.7 127 ~1 sodium phosphate Wash Eluate 69 145 10024 5.3 95.5 (=49 mleluate + 20 ml prefill

TABLE 9 Summary of conditions for Q Sepharose ™ HP step Flow rate VolumeFlow Step Buffer (cm/hr) (CV) Direction Regeneration 50 mM NaPi, 1M NaClpH 120 1 down 7.5 Equilibration 20 mM Tris-HCl, 10 mM 120 5 down NaCl pH7.5 Load Conditioned retentate step 4 120 down Wash 20 mM Tris-HCl, 10mM 120 5 down NaCl pH 7.5 Wash 20 mM NaPi, pH 7.5 120 1 down Elutioninto 50 mM NaPi, 0.2M NaCl, 120 4 down prefilled bag pH 7.5 Regeneration50 mM NaPi, 1M NaCl pH 120 3 down 7.5 CIP 1M NaOH 120 3 up Storage 10 mMNaOH 300 3 up

Example 7 Virus Reduction Step

Virus reduction may be performed at different stages of the process. Inthis example the virus reduction was performed after the polishing step.The eluate from the polishing step is nanofiltered, after 0.1 μmpre-filtration, through a Planova™ 15N filter. The following steps wereused:

-   -   The eluate from the polishing step is pre-filtered through a 0.1        μm filter. The filter is rinsed with a small volume of 50 mM        sodium phosphate, 0.2 M sodium chloride, pH 7.5 to remove        loosely bound product.    -   The eluate is filtered at pressure 0.8 bar, at room temperature.        The filtration is followed by a post wash of approximately three        Planova 15 system volumes with 50 mM sodium phosphate, 0.2 M        sodium chloride, pH 7.5.

Example 8 Formulation and Storage

Tangential flow filtration (TFF), with a 100 kDa, Screen A,polyethersulfone membrane (Sartorius™ or Millipore™) changes the bufferto formulation buffer. The tank can be cooled by a cooling jacket at +4°C. to keep the process at 21±5° C. The estimated capacity is 1001/m².The following steps were used for formulation and storage:

-   -   Equilibrate the membrane 3.5 mM Na₂HPO₄, 0.17 mM NaHPO₄, 250 mM        mannitol, 27 mM glycine, pH 7.7 (formulation buffer).    -   Dilute the purified product comprising alpha-mannosidase with        approximately 1 volume formulation buffer to target        concentration 2-3 mg/ml. If the protein concentration is low in        the product it is possible (but not necessary) to concentrate to        4-6 mg/ml to reduce the volume before dilution with formulation        buffer.    -   Concentrate ˜twice to target concentration 6 mg/ml by        ultrafiltration at TMP 0.8 and at 21±5° C.    -   Exchange 6 volumes by diafiltration against formulation buffer        at TMP 0.8, 21±5° C.    -   Concentrate ˜1.5 times by ultrafiltration and collect retentate.        Rinse the membrane with 1 system volume of formulation buffer to        remove loosely bound product. Collect the rinse together with        the retentate. An alternative is to measure OD 280 in the rinse        and pool only if it contains product. The final target protein        concentration is 7±2 mg/ml.    -   Clean the membrane with H₂O, followed by 0.5 M NaOH (60 min        contact time). Store in 0.1 M NaOH.

TABLE 11 Summary of conditions for the formulation TFF step UF/DF dil.Target conc. TMP Step Buffer factor (mg/ml) (bar) Comment Dilution ofFormulation 2 2-3 If protein conc. is step 6 buffer <4 mg/ml in step 6product product a UF step can be introduced before dilutionEquilibration Formulation buffer UF 2 6 ± 2 0.8 1.1 bar in/0.5 bar outDF Formulation 6 ± 2 6 ± 2 0.8 buffer UF ~1.5 7 ± 2 Collect retentateSystem wash Formulation 1 system 7 ± 2 if pooled Collect and pool withbuffer volume with retentate retentate if protein Rinse water CIP 0.5-1MNaOH store 0.1M NaOH

The product was diluted to 5 mg/ml and sterile filtered. The filtereddrug substance is filled into bottles and frozen.

Example 9 Fed Batch Cultivation Process for Alpha Mannosidase

After cell thaw, the cells were expanded in shake flasks, 10 L seedbioreactor and 50 L seed bioreactor before their transfer to aproduction bioreactor (250 L). At inoculation day of the productionbioreactor, the cell density in the 50 L seed bioreactor was between 2and 2.5 MVC/mL. The cells were inoculated in the production reactor fromthe seed bioreactor at a cell density of 0.5 MVC/mL so that the cellsuspension volume was 100 L when the inoculation was completed.Inoculation day is called day 0, the day after is called day 1, and soon. From day 1 to the end of the run, feed medium was added daily inboost according to a predefined rate (see below). From day 1 to the endof the run, glutamine and glucose were added daily in boost according topredefined rates and rules (see below). When the viable celldensity >2.2 MVC/mL or at day 3, whatever came first, the temperaturewas decreased to the production temperature

TABLE 12 Actual temperatures and experimental conditions. DO (%) 40 ± 5pH day 0 to day 2 (*) between 6.60 and 6.95 pH day 3 to end 6.9 ± 0.05Temperature (° C.) A) → shifted to B) when the A) 36.5 ± 0.5 temperatureshift condition is fulfilled → B) 31.0 ± 0.5 Temperature shift conditionCv ≧2.2 MVC/mL or day 3 (whatever comes first) Agitation rate (rpm) tobe adjusted according previous suggestion: 45 rpm, shear stressexperience of the production bioreactor tolerance of the cells supposedto be normal for CHO cells at 31° C. (not tested) Inoculation viablecell density, (MVC/mL), at day 0 0.5 ± 0.1 Max pCO₂ in liquid phaselevel not known, tentatively 18 kPa Feed of feed medium, glutamine andglucose See text below Glucose target in culture (mM) 6 [5.5 9.0]Glutamine target in culture (mM) 2 mM D1-D3, 1 mM D4 and forward Harvestcondition day 18-21 or viability <65%, whatever comes first Workingvolume (L): initial → final 100 → about 200 Alkali added for pH control0.5M Na₂CO₃ Dilution of inoculate cell broth from seed bioreactor to  4production bioreactor after completion of inoculation larger thanCriteria of the expanded cells in the seed bioreactor at the inoculationof the production bioreactor Minimal cell density (MVC/mL)  2.0 Maximalcell density (MVC/mL)  2.5 Number of millions viable cells 44 to 56Viability ≧93% Batch cultivation in seed bioreactor 50 L before 76inoculation of production bioreactor not longer than (hours) (*) Betweenday 0 and day 2, only CO₂ was automatically added by pH control. Alkaliwas not added unless pH would be <6.60.

Base Medium

ACF medium (ExCell302, SAFC) supplemented with 2 mM glutamine andcontaining 11.1 mM (2 g/L) glucose. The base medium without glutaminecan be transferred into the production bioreactor up to 3 days andstored at 36.5° C. i.e. during the sterility test of the bioreactor.Otherwise, the medium is stored at 4° C.

Feed Media The feed medium, E35, was 35% CHO CD Efficient Feed B(InVitrogen cat nr. SKU# A10240-01) feed concentrate diluted in ACFmedium without glutamine and containing 11.1 mM glucose. Feed medium wasstored dark at 4° C. The feed medium can stand dark during up to 96hours, i.e. four days, at room temperature during the cultivation.

TABLE 13 Feed medium volume added per day Days 0 1 to 5 6 to 16 Volumeadded/day in (L) 0 12 4

Additives

a) Stock solution of 2500 mM glucose. b) Stock solution of 200 mMglutamine. c) Alkali 0.5 M Na₂CO₃.

Delivery of Feed Medium, Glucose and Glutamine

Feed medium was pumped daily in boost. Glucose and glutamine were addeddaily to maintain their concentrations within the given targets byadding, in boost, stock solutions of glucose and glutamine in amountsaccording to below.

Glutamine Addition Rules:

-   -   Day 1 to day 3: add a volume of glutamine stock solution so that        the glutamine concentration in the bioreactor is 2 mM.    -   From day 4: add a volume of glutamine stock solution so that the        glutamine concentration in the bioreactor is 1 mM.

Glucose Addition Rules:

-   -   Day 0 to day 8: no addition of glucose stock solution    -   From day 9, if glucose concentration in production bioreactor ≦8        mM, add a volume of glucose stock solution so that the glucose        concentration in the bioreactor is 8 mM.

Process Overview:

TABLE 15 Day by day overview of cultivation process: Day Action −3 or −2Sterilization of the bioreactor and filling with ACF medium without orglutamine in volume 75 L or minimal volume to cover the probes (in casethis is larger than 75 L) Calibration of DO probe 0 a) Cell count ofexpanded cells in exponential growth from 50 L seed bioreactor andfulfilment of criteria for inoculation b) Removal of ACF medium fromproduction bioreactor if volume is >75 L. Stabilization of medium inproduction bioreactor at pH and temperature set points during at last 45min and addition of glutamine to obtain a final glutamine concentrationof 2 mM in 100 L medium. The glutamine present in the cell broth fromthe 50 L seed bioreactor is not taken into account. c) Transfer of cellbroth from 50 L seed bioreactor to production bioreactor d) Adjustmentof volume to 100 L cell suspension in production bioreactor e)Stabilization for ≈1 hr (between 45′ and 2 hrs 30′) and sample for cellcount, pH and metabolism parameters 1 to 2 Cell count, sample, feed offeed medium, feed of glucose and glutamine pH controlled between 6.60and 6.95 with automatic addition of CO2 (avoid alkali addition) 3 Cellcount, sample, feed of feed medium, feed of glucose and glutamine pHcontrolled at set point 6.9 ± 0.05 with automatic addition of alkali orCO₂ Decrease of temperature to 31° C. when either: 1) The viable celldensity has reached 2.2 MVC/mL or 2) On day 3  4 to 17 Cell count,sample, feed of respective feed media, feed of glucose and glutamine18-21 or Harvest viability <65%

Example 10 Characterization of the Purified Product ComprisingAlpha-Mannosidase

Table 16 below shows how the purification scheme of the presentinvention provided alpha-mannosidase with a high proportion of the 130kDa glycoprotein species, as compared to the breakdown products of 75and 55 kDa respectively. It is also shown how a 250 L process using a4-step purification process with improved wash steps for both thecapture step comprising multimodal ligands and the intermediate activestep as well as a Q sepharose HP polishing step performed better thanthe 30 L process using a 3 step process with respect to both yield,overall purity and yield of the 130 kDa species.

TABLE 16 Yields of alpha-mannosidase species in purified productPercentages of individual alpha- mannosidase species Purification Scale,method after purification and purity 130 kDa 55 kDa 75 kDa 250|4-step,70% yield, purity = 95.2% 1.5%  2.9% 99.6% 30|3-step, 60-70% yield,purity = 92.1% 2.6% 3.52% 98.2%

The distribution of species is seen in HPLC diagram of FIG. 7 for threeprocesses, where the above represent the 4-step and 3-step processesrespectively. The 2-step process shown is without using a multimodalligand step. The first peak from the left is the 55 kDa species,followed by the 130 kDa and 75 kDa species respectively.

REFERENCES

-   Hirsch et al. EMBO J. 22, 1036-1046, 2003 and Saint-Pol et al. J.    Biol. Chem. 274, 13547-13555, 1999-   Aronson and Kuranda FASEB J 3:2615-2622. 1989-   Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997-   Kaneda et al. Chromosoma 95:8-12. 1987-   Riise et al. Genomics 42:200-207, 1997-   Nilssen et al. Hum. Mol. Genet. 6, 717-726. 1997-   Liao et al. J. Biol. Chem. 271, 28348-28358. 1996-   Nebes et al. Biochem. Biophys. Res. Commun. 200, 239-245. 1994-   Chester et al., In: Durand P, O'Brian 3 (eds) Genetic errors of    glycoprotein metabolism. Edi-Ermes, Milan, pp 89-120. 1982-   Thomas and Beaudet. In: Scriver C R, Beaudet A L, Sly W A, Valle D    (eds) The metabolic and molecular bases of inherited disease. Vol 5.    McGraw-Hill, New York, pp 2529-2562. 1995-   Hocking et al. Biochem J 128:69-78. 1972-   Walkley et al. Proc. Nat. Acad. Sci. 91: 2970-2974, 1994-   Crawley et al. Pediatr Res 46: 501-509, 1999-   Stinchi et al. Hum Mol Genet 8: 1366-72, 1999-   Berg et al. Biochem J. 328:863-870. 1997-   Tollersrud et al. Eur J Biochem 246:410-419. 1997-   Walkley et al. Proc. Nat. Acad. Sci. 91: 2970-2974, 1994-   Will et al. Arch Dis Child 1987 October; 62(10):1044-9-   Barton et al. N Engl J Med 324:1464-1470-   Prows et al. Am J Med Genet 71:16-21-   Neufeld, E. F. Enzyme replacement therapy, in “Lysosomal disorders    of the brain” (Platt, F. M. Walkley, S. V: eds Oxford University    Press).-   Grubb et al. PNAS 2008, 105(7) pp. 2616-2621-   Roces et al. Human Molecular Genetics 2004, 13(18) pp. 1979-1988-   Blanz et al. Human Molecular Genetics 2008, 17(22) pp. 3437-3445-   WO 02/099092-   WO 05/094874-   WO 05/077093-   Berg et al. Molecular Genetics and Metabolism, 73, pp 18-29, 2001-   Heikinheimo et al. J. Mol. Biol. 327, 631-644, 2003-   Hansen et al., Biochem. J. (2004), 381, pp. 537-567

1. (canceled)
 2. A composition comprising alpha-mannosidase obtainableby a process for purification of a recombinant human lysosomalalpha-mannosidase from a cell culture, wherein a fraction of said cellculture comprising said recombinant human lysosomal alpha-mannosidase issubjected to chromatography on a resin comprising a multi-modal ligand,wherein said resin bound multi-modal ligand is a substance having acarboxylic acid or sulphonic acid group.
 3. A process for fed batch orcontinuous production of recombinant alpha-mannosidase, comprising thefollowing steps: a. inoculating a production reactor comprising a basemedium with cells capable of producing recombinant alpha-mannosidase onday 0, to provide a cell culture; b. adding a feed medium to said cellculture at least once from day 1; c. adjusting the temperature of saidcell culture to at the most 35° C., either after day 3 or when theviable cell density is higher than 2.1 MVC/mL, whichever comes first;and d. applying a process for purification of the recombinant humanlysosomal alpha-mannosidase from the cell culture, wherein a fraction ofsaid cell culture comprising said recombinant human lysosomalalpha-mannosidase is subjected to chromatography on a resin comprising amulti-modal ligand, wherein said resin bound multi-modal ligand is asubstance having a carboxylic acid or sulphonic acid group.
 4. Theprocess according to claim 3, wherein the cell culture is free of anysupplements derived from animals.
 5. The process according to claim 3,wherein the process for fed batch or continuous production is performedat a volume of at least 30 L.
 6. The process according to claim 3,wherein the recombinant human lysosomal alpha-mannosidase has a sequenceselected from the group consisting of: A) the sequence set forth in SEQID NO 2, and B) a sequence having at least 90% sequence identity to SEQID NO 2, wherein said sequence has lysosomal alpha-mannosidase activity.7. The composition comprising alpha-mannosidase obtainable by theproduction process according to claim
 3. 8. A composition comprisingpurified recombinant alpha-mannosidase, wherein at least 80% of thealpha-mannosidase is present as a 130 kDa glycoprotein.
 9. Thecomposition according to claim 8, wherein the recombinantalpha-mannosidase remains stable in liquid solution for at least 4 dayswhen stored at +5° C. or for at least 24 months when stored at −20° C.10. The composition according to claim 8, wherein the recombinant humanlysosomal alpha-mannosidase has a sequence selected from the groupconsisting of: A) the sequence set forth in SEQ ID NO 2, and B) asequence having at least 90% sequence identity to SEQ ID NO 2, whereinsaid sequence has lysosomal alpha-mannosidase activity.
 11. A method oftreating alpha-mannosidosis and/or reducing or alleviating the symptomsassociated with alpha-mannosidosis, said method comprising a step ofadministering a composition comprising: A) a composition comprisingalpha-mannosidase obtainable by a process for purification of arecombinant human lysosomal alpha-mannosidase from a cell culture,wherein a fraction of said cell culture comprising said recombinanthuman lysosomal alpha-mannosidase is subjected to chromatography on aresin comprising a multi-modal ligand, wherein said resin boundmulti-modal ligand is a substance having a carboxylic acid or sulphonicacid group; B) a composition comprising alpha-mannosidase obtainable bya process for fed batch or continuous production of recombinantalpha-mannosidase, comprising the following steps: i. inoculating aproduction reactor comprising a base medium with cells capable ofproducing recombinant alpha-mannosidase on day 0, to provide a cellculture; ii. adding a feed medium to said cell culture at least oncefrom day 1; iii. adjusting the temperature of said cell culture to atthe most 35° C., either after day 3 or when the viable cell density ishigher than 2.1 MVC/mL, whichever comes first; and iv. applying aprocess for purification of the recombinant human lysosomalalpha-mannosidase from the cell culture, wherein a fraction of said cellculture comprising said recombinant human lysosomal alpha-mannosidase issubjected to chromatography on a resin comprising a multi-modal ligand,wherein said resin bound multi-modal ligand is a substance having acarboxylic acid or sulphonic acid group; or C) a composition comprisingpurified recombinant alpha-mannosidase, wherein at least 80% of thealpha-mannosidase is present as a 130 kDa glycoprotein.